A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress.

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

Dicer prevents genome instability in response to replication stress.

Dicer, an endoribonuclease best-known for its role in microRNA biogenesis and RNA interference pathway, has been shown to play a role in the DNA damage response and repair of double-stranded DNA breaks (DSBs) in mammalian cells. However, it remains unknown whether Dicer is also important to preserve genome integrity upon replication stress. To address this question, we focused our study on common fragile sites (CFSs), which are susceptible to breakage after replication stress. We show that inhibition of the Dicer pathway leads to an increase in CFS expression upon induction of replication stress and to an accumulation of 53BP1 nuclear bodies, indicating transmission of replication-associated damage. We also show that in absence of a functional Dicer or Drosha, the assembly into nuclear foci of the Fanconi anemia (FA) protein FANCD2 and of the replication and checkpoint factor TopBP1 in response to replication stress is impaired, and the activation of the S-phase checkpoint is defective. Based on these results, we propose that Dicer pre-vents genomic instability after replication stress, by allowing the proper recruitment to stalled forks of proteins that are necessary to maintain replication fork stability and activate the S-phase checkpoint, thus limiting cells from proceeding into mitosis with under-replicated DNA.

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer.

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.

Tax impairs DNA replication forks and increases DNA breaks in specific oncogenic genome regions.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus associated with adult T-cell leukemia (ATL), an aggressive CD4 T-cell proliferative disease with dismal prognosis. The long latency preceding the development of the disease and the low incidence suggests that the virus itself is not sufficient for transformation and that genetic defects are required to create a permissive environment for leukemia. In fact, ATL cells are characterized by profound genetic modifications including structural and numerical chromosome alterations. RESULTS: In this study we used molecular combing techniques to study the effect of the oncoprotein Tax on DNA replication. We found that replication forks have difficulties replicating complex DNA, fork progression is slower, and they pause or stall more frequently in the presence of Tax expression. Our results also show that Tax-associated replication defects are partially compensated by an increase in the firing of back-up origins. Consistent with these effects of Tax on DNA replication, an increase in double strand DNA breaks (DDSB) was seen in Tax expressing cells. Tax-mediated increases in DDSBs were associated with the ability of Tax to activate NF-kB and to stimulate intracellular nitric oxide production. We also demonstrated a reduced expression of human translesion synthesis (TLS) DNA polymerases Pol-H and Pol-K in HTLV-I-transformed T cells and ATL cells. This was associated with an increase in DNA breaks induced by Tax at specific genome regions, such as the c-Myc and the Bcl-2 major breakpoints. Consistent with the notion that the non-homologous end joining (NHEJ) pathway is hyperactive in HTLV-I-transformed cells, we found that inhibition of the NHEJ pathway induces significant killing of HTLV-I transformed cells and patient-derived leukemic ATL cells. CONCLUSION: Our results suggest that, replication problems increase genetic instability in HTLV-I-transformed cells. As a result, abuse of NHEJ and a defective homologous repair (HR) DNA repair pathway can be targeted as a new therapeutic approach for the treatment of adult T-cell leukemia.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil­based chemotherapy.

BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil­based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II­III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II­III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012­0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages II­III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy.

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.

New Topoisomerase I mutations are associated with resistance to camptothecin.

BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

Does interference between replication and transcription contribute to genomic instability in cancer cells?

We have recently reported that topoisomerase 1 (Top1) cooperates with ASF/SF2, a splicing factor of the SR family, to prevent unscheduled replication fork arrest and genomic instability in human cells. Our results suggest that Top1 execute this function by suppressing the formation of DNA-RNA hybrids during transcription, these so-called R-loops interfering with the progression of replication forks. Using ChIP-chip, we have shown that γ-H2AX, a marker of DNA damage, accumulates at gene-rich regions of the genome in Top1-deficient cells. This is best illustrated at histone genes, which are highly expressed during S phase and display discrete γ-H2AX peaks on ChIP-chip profiles. Here, we show that these γ-H2AX domains are different from those induced by camptothecin, a Top1 inhibitor inducing double-strand DNA breaks throughout the genome. These data support the view that R-loops promote genomic instability at specific sites by blocking fork progression and inducing chromosome breaks. Whether this type of transcription-dependent fork arrest contributes to the replication stress observed in precancerous lesions is an important question that deserves further attention.

Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription.

Topoisomerase I (Top1) is a key enzyme in functioning at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing a conflict between transcription and DNA replication. Using DNA combing and ChIP (chromatin immunoprecipitation)-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase, and that breaks occur preferentially at gene-rich regions of the genome. Notably, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2), which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during the early stages of tumorigenesis.

Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis.

The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-kappaB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms.

Apoptosis regulation in tetraploid cancer cells.

Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and gamma-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells.

NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.

Selective resistance of tetraploid cancer cells against DNA damage-induced apoptosis.

Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system.

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability.

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.

Induction of multiple double-strand breaks within an hsr by meganucleaseI-SceI expression or fragile site activation leads to formation of double minutes and other chromosomal rearrangements.

Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceI-induced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.